Previous studies from Dr. Finley's laboratory defined the 17 subunits of the yeast proteasome regulatory particle. These samples had been exposed to high salt during conventional purification. Recently an affinity-purification method for proteasomes was developed by Dr. Finley's laboratory. Proteins from the salt wash fraction of the purification were identified by mass spectrometry and subsequently shown to be bona fide proteasome-associated proteins (PAPs). These appear to be novel in vivo components of the proteasome; some appear to be approximately stoichiometric. The new components are a ubiquitin-protein ligase (Hul5), a deubiquitinating enzyme (Ubp6), a protein that stabilizes the association of the proteasome core particle and the regulatory particle (Ecru29), and a protein that has been implicated in DNA repair (Bkn3). Each of these four genes is co-regulated with genes for known proteasome subunits, and strongly induced by the alkylating agent MMS. In addition, the known PAP proteins are all evolutionarily conserved. Dr. Finley proposes to define the complete set of major PAPs, and to study the roles of these proteins in proteasome function in vivo and in vitro. Sequences responsible for proteasome binding will be mapped within each PAP, and the phenotypic consequences of the loss of proteasome association will be determined. The spectrum of proteasome substrates stabilized by loss of function mutations in PAP genes will be assessed globally using powerful new mass spectrometry methods. Loosely associated cofactors are critical for the functioning of many protein complexes, such as polymerases, ribosomes, microtubules, and nuclear pores. Therefore, systematic investigation of proteasome-associated proteins may significantly enhance our understanding of this important protein complex. Specific Aims are as follows: Aim 1. To define the major proteasome-associated proteins. Aim 2. To search globally for degradation defects in PAP mutants by mass spectrometry. Aim 3. To localize within the proteasome the binding sites for specific associated proteins. Aim 4. To map sequences within proteasome-associated proteins that mediate their binding to proteasomes. Aim 5. To study the functions of specific proteasome-associated proteins.